The present disclosure relates generally to the biosynthesis of steviol glycosides. In particular, the present disclosure relates to a recombinant polypeptide that catalyzes the production of steviol glycosides such as rebaudioside D, rebaudioside E and a novel rebaudioside (rebaudioside Z).
Steviol glycosides are natural products isolated from Stevia rebaudiana leaves, and are widely used as high intensity, low-calorie sweeteners. Naturally occurring steviol glycosides have the same base structure (steviol) and differ in the content of carbohydrate residues (e.g. glucose, rhamnose, and xylose residues) at the C13 and C19 positions. Steviol glycosides with known structure include stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, and dulcoside A.
On dry weight basis, stevioside, rebaudioside A, rebaudioside C, and dulcoside A, account for 9.1, 3.8, 0.6, and 0.3% of the total weight of the steviol glycosides in the leaves, respectively, while the other steviol glucosides are present in much lower amounts. Extracts from Stevia rebaudiana plant are commercially available, which typically contain stevioside and rebaudioside A as primary compounds. The other steviol glycosides typically are present in the stevia extract as minor components. For example, the amount of rebaudioside A in commercial preparations can vary from about 20% to more than 90% of the total steviol glycoside content, while the amount of rebaudioside B can be about 1-2%, the amount of rebaudioside C can be about 7-15%, and the amount of rebaudioside D can be about 2% of the total steviol glycosides.
As natural sweeteners, different steviol glucosides have different degrees of sweetness and after-taste. The sweetness of steviol glycosides is significantly higher than that of sucrose. For example, stevioside is 100-150 times sweeter than sucrose with bitter after-taste, while rebaudioside A and E are 250-450 times sweeter than sucrose and the after-taste is much better than stevioside. Accordingly, the taste profile of any stevia extract is profoundly affected by the relative content of the steviol glycosides in the extract, which in turn may be affected by the source of the plant, the environmental factors (such as soil content and climate), and the extraction process. In particular, variations of the extraction conditions can lead to inconsistent compositions of the steviol glycosides in the stevia extracts, such that the taste profile varies among different batches of extraction products. The taste profile of stevia extracts also can be affected by plant-derived contaminants (such as pigments, lipids, proteins, phenolics, and saccharides) that remain in the product after the extractions process. These contaminants typically have off-flavors undesirable for the use of the stevia extract as a sweetener.
The majority of the steviol glycosides are formed by several glycosylation reactions of steviol, which typically are catalyzed by the UDP-glycosyltransferases (UGTs) using uridine 5′-diphosphoglucose (UDP-glucose) as a donor of the sugar moiety. In plants, UGTs are a very divergent group of enzymes that transfer a glucose residue from UDP-glucose to steviol. Stevioside is an intermediate in the biosynthesis of rebaudioside compounds. For example, glycosylation of the C-3′ of the C-13-O-glucose of stevioside yields rebaudioside A; and glycosylation of the C-2′ of the 19-O-glucose of the stevioside yields rebaudioside E. Further glycosylation of rebaudioside A (at 19-O-glucose) or rebaudioside E (at C-13-O-glucose) produces rebaudioside D. (FIGS. 1A-1C).
A practical approach to improve the taste quality of stevia extract is to increase the yield of rebaudioside compounds by further glycosylation of stevioside. The UGTs with a 1,2-19-O-glucose glycosylation activity are important enzymes for rebaudioside D and E production.
Sucrose synthases (SUS) catalyze the conversion of the UDP to UDP-glucose in the presence of sucrose. Thus, for a glycosylation reaction utilizing UDP-glucose (such as those catalyzed by the UGTs), SUS can be used to re-generate UDP-glucose from UDP, enhancing the efficiency of such reaction (FIG. 2).
Accordingly, there is a need for steviol glycosides with consistent taste profile and less off-flavor than the existing commercial products. As described herein, the present disclosure provides a recombinant polypeptide that is useful for preparing steviol glycosides (such as rebaudioside D and rebaudioside E). The present disclosure also provides a method of producing a steviol glycoside (rebaudioside Z) composition using such recombinant polypeptide.